Health food useful for preventing liver and biliary dysfunctions containing an alkanoyl l-carnitine and silybum marianum extract

ABSTRACT

A health food/dietary supplement is disclosed endowed with a protective action on liver function against exogenous and endogenous hepatotoxic agents the characterizing components of which are isovaleryl L-carnitine and/or propionyl L-carnitine and an extract of  Silybum marianum  (milk thistle) standardised to at least 70% by weight of silymarin.

[0001] The present invention relates to a health food/dietary supplementcontaining as its characterising components an alkanoyl L-carnitineselected from the group comprising isovaleryl L-carnitine and propionylL-carnitine or their pharmacologically acceptable salts or mixturesthereof and an extract of Silybum marianum (milk thistle) or its activecomponents, among which most notably silymarin.

[0002] It has been found that the above-mentioned composition isextremely effective in exerting a potent protective action on liverfunction and integrity against lesions induced by various exogenous andendogenous hepatotoxic agents owing to the unexpected synergistic effectexerted by the interaction of its components.

[0003] Isovaleryl L-carnitine, a natural component of the carnitinepool, presents a specific activity at lysosomal level and on thecytosolic movements of calcium. It is therefore capable of interveningin proteolytic processes such as occur during intense and prolongedeffort, and of -protecting a number of organs such as the liver againstthe action of toxic substances.

[0004] Propionyl L-carnitine exerts an intense antioxidant effect and isparticularly effective in improving the peripheral circulation andcardiac function.

[0005] In addition, muscular carnitine transferase possesses higheraffinity for propionyl L-carnitine than for L-carnitine, andconsequently propionyl L-carnitine possesses a higher degree ofspecificity for cardiac and skeletal muscle. Moreover, propionylL-carnitine transferase by transporting the propionyl group increasesthe uptake of this component by the muscle cells, which may beparticularly important for energy purposes, in that the propionate canbe used by the mitochondria as an anapleurotic substrate and supplyenergy in the absence of oxygen.

[0006] The milk thistle is an indigenous plant cultivated for centuriesin the Mediterranean region and South Western Europe and naturalised inmost of Europe. It is also naturalised in North America, particularlyCalifornia; in South America it is present from Uruguay to Chile andEcuador; in Australia it runs wild; it is commonly found in abandonedfields, old pasture land and at the roadside.

[0007] The efficacy of the seeds of Silybum marianum or the milk thistlein preventing or treating various forms of liver or biliary dysfunctionhas been known for over two thousand years and is deeply rooted inpopular tradition.

[0008] In 1968, silymarin, a flavanolignan complex was isolated from theplant. This consists mainly of three flavanolignans, silybin(silybinin), silychristin and silydianin. Other flavanolignans presentare: dehydrosilybin, 3-deoxysilychristin, deoxysilydianin (silymonin),silyandrin, silybinome, silyermin and neosilyermin. Other constituentsare apigeninin and silybonol; a fixed oil (16-18%), composed mainly ofoleic acid and linoleic acid, in addition to myristic, palmitic andstearic acids.

[0009] In recent decades, few principles of vegetable origin have beenstudied as thoroughly as silymarin.

[0010] Its main activity is liver-protective and antioxidant. Theliver-protective activity of silymarin has been demonstrated in numerousexperimental models in which liver damage is induced by toxicsubstances, including carbon tetrachloride, galactosamine,thioacetamide, hepatotoxic cold-blooded frog virus, lanthanides and thetoxins of Amanita phalloides, phalloidin and α-amanitin.

[0011] The liver-protective efficacy is based on various distinctmechanisms of action. Silymarin stimulates RNA polymerase A, increasingribosomal protein synthesis and causing activation of the regenerativecapacity of the liver through cell development. Silymarin interacts withthe membranes of liver cells, blocking the binding sites and preventingintake of toxins, as demonstrated in rabbit liver microsomes and inmononuclear lipid layers. It is strongly antioxidative (free radicalscavenger activity 10 times greater than that of Vitamin E), in that itblocks the release of malonyldialdehyde and antiperoxidative activity.

[0012] Clinical trials have suggested that preliminary treatments withsilymarin inhibit the liver damage induced by alcohol, industrialchemicals and psychotrope drugs, accelerating the normalisation ofimpaired liver functions.

[0013] The patients receiving silymarin show a rapid improvement inelevated serum levels of glutamic-oxalacetic transaminase (GOT),glutamic-pyruvic transaminase (GPT) and γ-glutamyl transpeptidase(γ-GT).

[0014] The therapeutic activity of silymarin as a drug is extensivelydocumented against liver damage, in chronic inflammation supporttreatment and in cirrhosis, including chronic hepatitis and fattyinfiltration of the liver caused by alcohol and other chemicals. For thepurposes of infusions, silbinin preparations are used for supporttreatment in cases of poisoning due to the toadstool Amanita.

[0015] In the herbalist or food product sector, the seeds of the plantor an extract of the seeds are used in tea, capsules, tablets and herbteas, mainly as a liver detoxifier.

[0016] The commercial preparations of milk thistle include ethanolextracts of seeds and/or fruits, pills or capsules (35-70 mg)standardised to 70% silymarin, in average daily doses of 200-400 mg.

[0017] It has now surprisingly been found that a composition containingas its characterising components a combination of:

[0018] (a) an alkanoyl L-carnitine selected from the group consisting ofisovaleryl L-carnitine, propionyl L-carnitine or their pharmacologicallyacceptable salts or mixtures of the same; and

[0019] (b) an extract of Silybum marianum standardised to at least 70%silymarin by weight,

[0020] constitutes an effective health food/dietary supplement exertinga potent protective action on liver function and integrity againstlesions induced by various exogenous and endogenous hepatotoxic agentsowing to the potent and unexpected synergistic effect exerted by itscomponents.

[0021] The dietary supplement described in this invention mayadditionally contain

[0022] (c) a “carnitine” selected from the group comprising L-carnitine,acetyl L-carnitine, butyryl L-carnitine and valeryl L-carnitine, ortheir pharmacologically acceptable salts or mixtures thereof.

[0023] The weight-to-weight ratios of the above-mentioned components(a):(b):(c) range from 1:0.1:1 to 1:1:2.

[0024] The surprising synergistic effect which is achieved with thecombination of “carnitines” (a term used to indicate collectively bothL-carnitine and the alkanoyl L-carnitines), particularly isovalerylL-carnitine and/or propionyl L-carnitine and the Silybum marianumextract, has been demonstrated by various pharmacological tests (some ofwhich are described here below) chosen in such a way as to be stronglypredictive of the practical use of this composition in the preventive,nutritional and dietetic fields.

[0025] Tests of protection against toxic effects induced by carbontetrachloride on isolated liver cell cultures

[0026] In these tests, rat liver cells were isolated according to themethod described by Segler (Segler P. O., Methods Cell. Biol. Chem.,264, 4747, 1989) and were used to determine the lipoperoxidative andtoxic effects induced by CCl₄ (100 mg L⁻¹) measuring, in the supernatantliquid of the cell culture, the two markers, alanine-aminotransferase(AlaAT) and aspartate aminotransferase (AspAT) (Auto-biochemistry AssaySystem-Beckman 700 - Encore-2), and malonaldehyde, as a peroxidationindicator, using the thiobarbituric acid method (Ohka W. H., Anal.Biochem., 95, 351, 1979).

[0027] The protective effect was then measured against CCl₄ toxicity byintroducing into the liver cell cultures both isovaleryl L-carnitine(100 mg L⁻¹) and propionyl L-carnitine (100 mg L⁻¹) or silymarin (50 mgL⁻¹) or a combination of these products at the same doses. The cytologyof the liver cells submitted to this treatment was also observed afterfixation in formalin or glutaraldehyde, under both the optical andelectron microscopes.

[0028] Tables 1 and 2 give the results of these tests which show thatboth isovaleryl L-carnitine and propionyl L-carnitine are capable ofpartly inhibiting the toxicity induced by CCl₄, whereas the protectiveeffect exerted by silymarin is greater. Surprisingly effective is theprotection induced by the combination of the above-mentioned alkanoylL-carnitines and silymarin. In this case, in fact, the toxic andlipoperoxidative effects caused by CCl₄ on liver cell cultures arealmost totally inhibited.

[0029] This significant and surprising protective effect was alsodetected in the cytological examination carried out on the liver cells,which revealed a reduction in the necrotic cells present in the culture,but was detectable above all at ultrastructural examination. In thecontrol group (CCl₄), the cells showed heterochromatin abnormalities,disappearance of the mitochondrial crests in the mitochondria and areduction in the number of ribosomes. In those cells which, in additionto CCl₄, were also exposed to the alkanoyl L-carnitines and silymarin,the cell membrane and the nucleus appeared unexpectedly intact and boththe heterochromatins and mitochondria and the number of ribosornesappeared to be regular. TABLE 1 Protection against CCl₄ toxicity onliver cell cultures Concentration of AlaAT (nmol.min⁻¹.L⁻¹) insupernatant of liver cells exposed to CCl₄ (controls) together withisovaleryl L-carnitine (I), propionyl L-carnitine (P), and silymarin(S), alone or in combination Time (hours) Controls I P S I + S P + S 426.8 ± 3.2 19.4 ± 2.4 22.3 ± 4.1 20.2 ± 3.1 10.4 ± 1.7 12.6 ± 1.8 8 28.6± 2.2 16.7 ± 3.1 18.4 ± 2.3 18.8 ± 2.7 8.5 ± 3.1 9.8 ± 2.1 16 32.4 ± 5.115.6 ± 2.7 16.6 ± 1.9 16.2 ± 1.7 5.2 ± 1.7 6.6 ± 2.7

[0030] TABLE 2 Concentration of AspAT (nmol.min⁻¹.L⁻¹) in supernatant ofliver cells exposed to CCl₄ (controls) together with isovalerylL-carnitine (I), propionyl L-carnitine (P), and silymarin (S), alone orin combination Time (hours) Controls I P S I + S P + S 4  8.5 ± 0.6 6.9± 1.1 7.5 ± 0.9 6.4 ± 0.7 4.9 ± 0.8 5.1 ± 0.9 8 11.6 ± 0.1 6.2 ± 0.9 5.5± 1.2 5.1 ± 2.1 3.7 ± 0.5 4.0 ± 0.7 16 12.2 ± 1.1 5.5 ± 1.2 5.2 ± 1.44.9 ± 1.5 2.2 ± 0.8 2.5 ± 0.9

[0031] Tests of protection against hepatotoxic effects induced bygalactosamine in the rat

[0032] In these tests, the protective activity of isovaleryl L-carnitineagainst galactosamine-induced liver intoxication was confirmed (ZezzaF., Pharmacol. Res., 27, (Suppl. 1), 85, 1993). At the same time, apotent and surprising synergistic protective effect of the combinationof isovaleryl L-carnitine and silymarin was detected against suchintoxication, which presents characteristics similar to those of viralhepatitis.

[0033] In these tests the method adopted was that described by Reuter(Reuter W., Trends in the Therapy of Liver Diseases Proceeding, Ed. A.Bertelli, pp. 121, 1974, Karger Basel 1975).

[0034] Rats weighing approximately 250 g received daily oraladministrations for two days consecutively of 200 mg/kg of isovalerylL-carnitine or 200 mg/kg of propionyl L-carnitine or 100 mg/kg ofsilymarin or a combination of these compounds at the same doses. After24 hours, the same rats were administered 400 mg/kg i.p. ofgalactosamine. After a further 24 hours, blood samples taken from theanimals were used to determine both glutamic-oxalacetic transferase(GOT) and the bilirubin present in plasma, according to the methoddescribed by Keppler (Keppler D., Exp. Mol. Pathol, 9, 279, 1968). Boththe enzyme activity and bilirubin were reduced by the administration ofpropionyl L-carnitine or, to a greater extent, by isovaleryl L-carnitineand silymarin, but returned to levels close to normal with theadministration of the alkanoyl L-carnitine/silymarin combination. TABLE3 Galactosamine intoxication tests Treatment GOT GluDH BilirubinControls   25 ± 4  4.5 ± 0.4 0.12 ± 0.01 Galactosamine  750 ± 68  595 ±51 1.08 ± 0.51 Isovaleryl L-carnitine  215 ± 30.5   64 ± 6.8 0.15 ± 0.04Silymarin  125 ± 15.3   71 ± 5.5  0.14 ± 0.02 Isovaleryl L-carnitine +34.4 ± 5.2  5.8 ± 0.6 0.11 ± 0.03 silymarin Propionyl L-carnitine + 45.7± 4.4  6.5 ± 0.7 0.12 ± 0.05 silymarin

[0035] Some non-limiting examples of combination compositions accordingto the present invention are given hereinbelow: 1) Propionyl L-carnitine500 mg Extract of milk thistle containing 200 mg flavanolignansexpressed as silymarin 2) Isovaleryl L-carnitine 500 mg Extract of milkthistle containing 200 mg flavanolignans expressed as silymarin 3)Isovaleryl L-carnitine 150 mg Propionyl L-carnitine 150 mg AcetylL-carnitine 150 mg Extract of milk thistle containing 200 mgflavanolignans expressed as silymarin 4) Isovaleryl L-carnitine 400 mgExtract of milk thistle containing 100 mg flavanolignans expressed assilymarin Choline 50 mg Arginine 50 mg Lysine 50 mg Ornithine 25 mgInositol 50 mg Methionine 25 mg 5) Isovaleryl L-carnitine 400 mg Extractof milk thistle containing 100 mg flavanolignans expressed as silymarinExtract of Cynara scolimus (artichoke) 25 mg expressed as cynarine Vit.C 50 mg Vit. E 5 mg Vit. PP 25 mg Choline 50 mg Coenzime Q₁₀ 25 mgSelenomethionine 50 μg

[0036] What is meant by a pharmacologically acceptable salt of thevarious aforesaid carnitines mentioned in the present specification is,in addition to the respective “inner salts”, any salt of these with anacid which does not give rise to unwanted toxic or side effects. Theseacids are well known to pharmacologists and to experts in pharmaceuticaltechnology.

[0037] Non-limiting examples of such salts are the following: chloride;bromide; iodide; aspartate, acid aspartate; citrate, acid citrate;tartrate; phosphate, acid phosphate; fumarate, acid fumarate;glycerophosphate; glucose phosphate; lactate; maleate, acid maleate;mucate; orotate; oxalate, acid oxalate; sulphate, acid sulphate;trichloroacetate; trifluoroacetate and methane sulphonate.

[0038] Among these salts, isovaleryl L-carnitine acid fumarate (U.S.Pat. No. 5,227,518) is particularly preferred.

[0039] A list of FDA-approved pharmacologically acceptable acids isgiven in Int. J. Pharm., 33, 1986, 201-217, the latter publication beingincorporated in the present specification by reference.

[0040] The supplement of the invention may further comprise vitamins,coenzymes, mineral substances, aminoacids and antioxidants. Thesupplement may be manufactured in the form of tablets, lozenges,capsules, pills, granulates, syrups, herb teas, vials or drops.

1. A food/dietary supplement which comprises the followingcharacterizing ingredients: (a) an alkanoyl L-carnitine selected fromthe group comprising isovaleryl L-carnitine, propionyl L-carnitine or apharmacologically acceptable salts thereof or mixtures thereof; and (b)an extract of silybum marianum standardized to at least 70% by weight ofsilymarin.
 2. The supplement of claim 1, further comprising: (c) a“carnitine” selected from the group comprising L-carnitine, acetylL-carnitine, butyryl L-carnitine and valeryl L-carnitine or thepharmacologically acceptable salts or mixtures thereof.
 3. Thesupplement of anyone of the preceding claims which further comprisesvitamins, sugars, coenzymes, mineral substances, aminoacids, peptidesand antioxidants.
 4. The supplement of any of the preceding claimswherein the pharmacologically acceptable salt is selected from the groupcomprising: chloride; bromide; iodide; aspartate, acid aspartate;citrate, acid citrate; tartrate; phosphate, acid phosphate; fumarate,acid fumarate; glycerophosphate; glucose phosphate; lactate; maleate,acid maleate; mucate; orotate; oxalate; acid oxalate; sulphate, acidsulphate; trichloroacetate; trifluoroacetate and methane sulphonate. 5.The supplement of any of the preceding claims having protective actionon liver function and integrity against lesions induced by exogenous andendogenous hepatotoxic agents.
 6. The supplement of any of the precedingclaims manufactured in solid or liquid form.
 7. The supplement of any ofthe preceding claim in the form of tablets, capsules, lozenges, pills,granulates, syrups, herb teas, vials or drops.
 8. The supplement of anyof the preceding claims wherein the weight ratio of components(a):(b):(c) ranges from 1:0.1:1 to 1:1:2.
 9. The supplement of claim 8,in unit dosage form, comprising: Propionyl L-carnitine 500 mg Extract ofmilk thistle containing 200 mg flavanolignans expressed as silymarin


10. The supplement of claim 8, in unit dosage form, comprising:Isovaleryl L-carnitine 500 mg Extract of milk thistle containing 200 mgflavanolignans expressed as silymarin


11. The supplement of claim 8, in unit dosage form, comprising:Isovaleryl L-carnitine 150 mg Propionyl L-carnitine 150 mg AcetylL-carnitine 150 mg Extract of milk thistle containing 200 mgflavanolignans expressed as silymarin


12. The supplement of claim 8, in unit dosage form, comprising:Isovaleryl L-carnitine 400 mg Extract of milk thistle containing 100 mgflavanolignans expressed as silymarin Choline 50 mg Arginine 50 mgLysine 50 mg Ornithine 25 mg Inositol 50 mg Methionine 25 mg


13. The supplement of claim 8, in unit dosage form, comprising:Isovaleryl L-carnitine 400 mg Extract of milk thistle containing 100 mgflavanolignans expressed as silymarin Extract of Cynara scolimus(artichoke) 25 mg expressed as cynarine Vit. C 50 mg Vit. E 5 mg Vit. PP25 mg Choline 50 mg Coenzime Q₁₀ 25 mg Selenomethionine 50 μg


14. A method for preventing the damages induced by exposure to exogenousand endogenous hepatotoxic agents and exerting a protective action onliver function and integrity which comprises administering to anindividual in need thereof a combination composition comprising thefollowing ingredients: (a) an alkanoyl L-carnitine selected from thegroup comprising isovaleryl L-carnitine, propionyl L-carnitine or apharmacologically acceptable salts thereof or mixtures thereof; and (b)an extract of silybum marianum standardized to at least 70% by weight ofsilymarin.